1. Field of the Invention
The present invention relates to an artificial cartilage tissue prepared using a tissue engineering technique, and a production method thereof.
2. Description of Related Art
Articular cartilage is an important tissue which covers the osteoepiphysis and is in charge of the smooth movement of joints. The articular cartilage has a structure in which chondrocytes are dispersed throughout a non-cellular tissue called an extracellular matrix (hereunder, referred to as “matrix”) that is neither innervarated nor vascularized. Such a structure in which these cells are dispersed throughout a matrix, that is, the fact that a small number of cells exist per unit volume, and the absence of vasculature means a very poor ability of self-regeneration when an injury occurs.
When articular cartilage is damaged due to aging, trauma, or the like, smooth movement of the joint is interferred and an acute pain occurs in the joint. Although the cartilage itself is not innervated, since the articular capsule, which constitutes the joint, and the subchondral bone (bone under the cartilage) have sensory nerves, a feeling of pain is experienced.
If the cartilage tissue containing chondrocytes remains at the damaged site of the cartilage, there is a possibility of medicinal treatment by means of a growth factor, or the like. However, if the cartilage tissue itself is defective, there is no way to regenerate the cartilage other than implantation of external chondrocytes or cartilage tissue, or introduction of myeloid cells from the bone marrow underneath the cartilage so as to form a cartilage. The method of introducing myeloid cells from the bone marrow to form a cartilage is currently applied to traumatic cartilage damage. However, the regenerated cartilage is fibrocartilage which is poor in mechanical strength, and thus it is said that joint function lasts only for several years.
Moreover, it is difficult to introduce myeloid cells to form a cartilage at the cartilage defective site of osteoarthritis, which is a chronic degenerative disease of cartilage. The current state is that there is no way other than artificial joint replacement as a treatment method for the last stage of osteoarthritis. Artificial joint replacement is effective for relieving acute pains in the joint, but it is regarded as a “last resort” since the joint function is largely limited and the life span of the artificial joint is about 15 years (the interval between the bone of the patient and the artificial joint is loosened).
From the above reasons, treatment methods by means of implantation of chondrocytes or a cartilage tissue are drawing attention. As one of such methods, there is a means in which cartilage is collected from a dead body or the patient him/herself together with the bone underneath thereof (subchondral bone), and is grafted into the cartilage defective site. The method of using an osteochondral graft collected from a dead body involves problems of donor shortages and risks of infection. The method of using an osteochondral graft collected from a non weight-bearing region of the patient him/herself is called mosaicplasty, and recently, many clinical examples thereof have been reported. However, there are shortcomings in that the collection quantity is limited, and a large osteochondral defect wound remains at the collection site.
As another treatment method of implantating chondrocytes or cartilage tissue, there is a method of filling cultured chondrocytes or cultured cartilage tissue. This method is characterized in that a small amount of cartilage tissue is collected from the patient him/herself or someone else, and chondrocytes are isolated, then the chondrocytes are cultured in vitro until a target number of cells or a target shape is achieved. After a fixed period of culturing, the cultured chondrocytes or the cultured cartilage tissue (hereunder, a cartilage-repairing product) is implanted into the cartilage defective site. Such a technique is generally called a “regenerative medical technique” or “tissue engineering”, and is under consideration not only for cartilage but also for various tissues and organs. Because of progress in control techniques of cell differentiation, recently it is shown that cultured chondrocytes can be prepared from embryo-stem cells or mesenchymal stem cells.
Various forms are being considered for implantation of artificial cartilage tissue prepared by culturing. As a leading example, there is a method in which chondrocytes isolated from a patient are monolayer cultured so as to increase the number of cells, which are then implanted into the cartilage defective site as a cell suspension (for example, refer to N. Engl. J. Med. 1994, 331, 889-895 (hereunder, called “reference 1”)). In this method, in order to prevent the implant material from being scattered and lost, the implanted site is covered with a periosteal patch after the implantation.
As another method, a method is reported in which chondrocytes collected from a patient are inoculated in a scaffold (scaffolding material) of atelocollagen, and cultured, after which they are implanted as a paste-like collagen gel (for example, refer to Japanese Unexamined Patent Application, Publication No. 2001-224678 (hereunder, called “reference 2”)).
Furthermore, in addition to the abovementioned atelocollagen, bioabsorbable synthetic polymers such as lactic acid/glycolic acid copolymer (PLGA) are also used as a scaffold of chondrocytes. Since these synthetic polymers have a high initial strength and may be given an arbitrary three dimensional shape, they are used not only for articular cartilages but also for preparing cultured auricular cartilages (for example, refer to Plast. Reconstr. Surg. 1999, 103, 1111-1119 (hereunder, called “reference 3”)).
However, in the method of reference 1, if chondrocytes are monolayer cultured, inherent characteristics of the chondrocytes partially disappear (dedifferentiation), and therefore, requirements for the-implant material, such as “redifferentiation”, “biosynthesis of the matrix”, and “repair of the defective site” are increased, thus the time required for recovery is inevitably elongated. Moreover, a large number of the implanted cells are scattered and lost during that period, and thus many researchers are raising questions about the effect.
Moreover, in the method of reference 2, the implanted site is covered with a periosteal patch after implantation. This method minimizes dedifferentiation of chondrocytes, and thus better effects are expected compared to the monolayer cultured chondrocytes. However, a drawback is the necessity of the periosteal patch. Furthermore, because of the paste-like form, the strength is much weaker compared to the actual cartilage. This means that it takes a long time to form the actual cartilage, and it is difficult for it to be compatible with the cartilage of the weight-bearing region of the joint.
Furthermore, in the method of reference 3, when a synthetic polymer is used for articular cartilage, the engraftment rate after implantation is poor. The reason is that, since the cartilage is an avascular tissue without a bloodstream, it takes a long time for the synthetic polymer to disappear even if it is a bioabsorbable synthetic polymer, which may interfere with the fusion between the implant material and the surrounding tissue, and the alteration of the matrix composition into that of original articular cartilage during the time until the polymer disappears. In order to fundamentally solve the various problems involved in the usage of scaffolds, ideal is a cartilage-repairing product substantially free of scaffold.
On the other hand, serum or plasma used for culturing involves a very big problem in terms of the safety of the prepared artificial cartilage tissue. That is, many of the cultured chondrocytes or cultured cartilage tissues, reported so far, are prepared using fetal bovine serum (FBS). FBS shows a superior growth-stimulative effect and matrix synthesis-stimulative effect for chondrocytes, and thus is very effective for preparing artificial cartilage tissues, but has a risk of bovine spongiform encephalopathy (BSE) and a risk of immunogenicity due to a contaminant protein derived from FBS mixed in the artificial cartilage tissue. In the case of humans, such risks can be reduced by using an allogenic serum or plasma, however conversely, risks of human infections such as human immunodeficiency disease and hepatitis are increased. If no serum nor plasma is used, such risks can be completely excluded, but preparation of engraftable artificial cartilage tissues is very difficult in serum-free media or plasma-free media. Therefore, using cells derived from cartilage of the patient him/herself and an autologous serum or plasma is optimal in terms of ensuring safety, and is practical. However, it is difficult to prepare artificial cartilage tissues which are suturable with a superior engraftablility, that is, having a mechanical strength above a fixed level.
As described above, the fact is that artificial cartilage tissues have not been developed yet which, are substantially free of exogenous scaffold material and heterologous protein, have a superior mechanical strength, and can be expected to satisfactorily engraft. In terms of safety, an artificial cartilage tissue is preferable that is prepared using cells derived from an autologous cartilage and an autologous serum or plasma. Moreover, for implantation, an artificial cartilage tissue is desirable which can be sutured to a target site without using a periosteal patch, or the like.